The Government has rights in this invention pursuant to NIH Grants GM 33464 and NRSA GM 12274, awarded by the Department of Health and Human Services.
This invention relates to novel compounds which function as irreversible ligands for nonsteroidal antiinflammatory drug (NSAID) and prostaglandin binding sites.
Nonsteroidal antiinflammatory drugs are drugs of choice in the treatment of arthritis and other inflammatory disorders. It is widely accepted that NSAIDs inhibit the enzyme Prostaglandin H.sub.2 synthase (PGH synthase) and prevent the synthesis of the primary prostaglandins which mediate some symptoms of inflammation. Since its introduction into the pharmaceutical market in 1963, indomethacin (I) has become the standard to which all other NSAIDs are compared. ##STR1## Another class of compounds which are equipotent with the indole acetic acids in their ability to inhibit PGH synthase are the N-phenylanthranilic acids, represented by meclofenamic acid (II) and diclofenac (III). The pyrrole acetic acids, represented by tolmetin (IV) and zomepirac (V), are slightly less potent inhibitors of PGH synthase. ##STR2##
Although the putative target enzyme, PGH synthase, has been purified to homogeneity and its sequence deducted from its cDNA, no good topographical map of the antiinflammatory drug binding site of
PGH synthase currently exists. Until antiinflammatory drug-receptor interactions are explicitly understood at the molecular level, progress in the rational design of superior drugs is hindered
This issue is complicated by the fact that PGH synthase may not be the only target for NSAID action. Since this enzyme is not inhibited by sodium salicylate, a presumptive active ingredient of acetyl salicylate (aspirin), alternative targets for the action of NSAIDs have been proposed Prostaglandin dehydrogenases are a family of NAD(P).sup.+ -linked oxidoreductases that display some regio-and stereoselectivity for the oxidation of hydroxyprostaglandins. Several of these enzymes are inhibited in a reversible fashion by NSAIDs. Inhibition of prostaglandin transformation at this level may contribute to the mechanism of action of NSAIDs.
Another possible target enzyme is 3.alpha.-hydroxysteroid dehydrogenase (3.alpha.-HSD, E.C. 1.1.1.50). The homogeneous 3.alpha.-HSD of rat liver cytosol is an unusual oxidoreductase in that it will catalyze the interconversion of 3.alpha.- (axial) alcohols to ketones on the steroid nucleus, and will function as a 9, 11 and 15- hydroxyprostaglandin dehydrogenase. 3.alpha.-HSD is potently inhibited at its active site by all the major classes of NSAIDs. By virtue of its prostaglandin dehydrogenase activity, it may represent an alternative site for NSAID action. It fulfills many of the criteria expected of a target enzyme: the concentrations of drugs required to inhibit 3.alpha.-HSD are comparable to those required to inhibit PGH synthase; the rank order of inhibitory potency of NSAIDs for 3.alpha.-HSD correlates extremely well with the human dose required to produce their pharmacological effect; the enzyme can distinguish between active and inactive geometric isomers of NSAIDs. On this basis, a rapid spectrophotometric screen has been developed for the enzyme, which can be used to screen potential antiinflammatory drugs. In addition, 3.alpha.-HSD catalyzes a pro-inflammatory reaction (PGF.sub.2.alpha. to PGE.sub.2) which is blocked by NSAIDs. The indomethacin sensitive 3.alpha.-HSD is widely distributed in rat tissues, with the enzyme of highest specific activity being found in liver, then lung, testis, heart, prostate, spleen, seminal vesicle, and brain. Despite 3.alpha.-HSD's involvement in steroid hormone metabolism, the enzyme is not present in highest levels in those tissues traditionally associated with endocrine function, but is high in those that rapidly metabolize prostanoids (lung and heart). (For a Review see Steroids, 47, 221-247 (I986))
Since 3.alpha.-HSD is a putative target enzyme for NSAIDs, knowledge of the topography of the enzyme's antiinflammatory drug and prostaglandin binding site could aid in future drug design. This approach may offer distinct advantages over working with PGH synthase which is difficult to assay and purify in large quantities. By contrast, 3.alpha.-HSD can be assayed spectrophotometrically, it can be purified in milligram quantities (50 mgs/purification), it has been crystallized and is undergoing x-ray diffraction analysis, and its cDNA has been cloned and is currently being sequenced.
One method of mapping the antiinflammatory drug binding site of 3.alpha.-HSD is by synthesizing affinity labeling analogs of NSAIDs, i.e. acylating agents. Affinity labeling analogs of substrates and inhibitors of hydroxysteroid dehydrogenases (HSDs) have been widely used to characterize steroid binding sites.(J. Biol. Chem., 247, 3424-3433 (1972); J. Biol Chem., 250, 7656-7662 (1975)). Indeed, bromoacetoxy analogs of dihydrotestosterone and desoxycorticosterone have been used to characterize the steroid binding domain of 3.alpha.-HSD. In this instance, attack of radiolabeled bromoacetoxy steroids by an enzyme nucleophile leads to the formation of covalently modified enzyme. Upon complete acid hydrolysis of the inactivated 3.alpha.-HSD, the steroid is released as the free alcohol, leaving behind a radiolabeled carboxymethylated amino acid which was identified as a carboxymethyl cysteine from its elution position on an amino acid analyzer. Furthermore, enzymatic digestion of the inactivated radiolabeled enzyme has lead to the purification and partial sequence of active site peptides.
In a similar study, mesylate analogs of dexamethasone have been used to identify amino acids and peptides involved in steroid binding to the glucocorticoid receptor (J. Biol. Chem., 262, 9669-9675, (1987); J. Biol. Chem., 263, 6842-6846 (1988)).
The use of affinity labeling analogs as inhibitors of HSD's and steroid receptors has proven useful in understanding the topography of these steroid binding sites. Application of this method to the NSAID and prostaglandin binding sites of 3.alpha.-HSD, prostaglandin dehydrogenases, and PGH synthase would be expected to reveal similar information and aid in the understanding of binding of NSAIDs to these sites.
Prostaglandin H.sub.2 Synthase (PGH synthase, E.C. 1.14.99.1) is a dual function enzyme, catalyzing the cyclooxygenation of arachidonic acid to PGG.sub.2, and its further peroxidation to PGH.sub.2 (Scheme A). It has been studied in a variety of tissues, but the seminal vesicles have been typically used for kinetic analyses NSAIDs are known to inhibit only the cyclooxygenase portion of the enzyme's activity, and it is this function which has been attributed to their overall effect. PGH synthase has been purified to homogeneity from ovine and bovine seminal vesicles and the amino acid sequence deduced from its cloned cDNA. Extensive kinetic characterization has shown that the enzyme can catalyze its self destruction in the presence of its substrates (arachidonic acid and O.sub.2), presumably by a free radical mechanism. Kinetic studies of the inhibition of PGH synthase by NSAIDs have suggested that although these drugs are competitive with arachidonic acid, they may also bind to a second site. NSAIDs are also capable of promoting self destruction of PGH synthase, although a covalent complex has never been isolated. In contrast, the methyl esters of NSAIDs bind reversibly to the PGH synthase active site and do not promote self destruction of the enzyme. Despite these studies, the topography of the NSAID binding site has remained poorly defined. Through the use of affinity labeling analogs of NSAIDs described herein, amino acids involved in NSAID binding could be identified, active site peptides could be purified and sequenced, and the position of the active site could be deduced from the sequence of the cDNA clone. This knowledge would greatly aid in the future design of NSAIDs. ##STR3##
In addition to PGH synthase, indomethacin and other NSAIDs have been shown to inhibit at low concentrations several prostaglandin dehydrogenases. They inhibit the action of 15-hydroxyprostaglandin dehydrogenase involved in the conversion of PGE.sub.2 to 15-keto-PGE.sub.2. NSAIDs also inhibit 13,14-prostaglandin reductase and 9-hydroxyprostaglandin dehydrogenase activities. Affinity labeling analogs based on NSAIDs have the capability of mapping the topography of the active site of these enzymes.
Affinity labeling analogs of NSAIDs also have the potential to act as ligands for affinity chromatography and would aid in the purification of PGH synthase, prostaglandin dehydrogenases and NSAID receptors and binding sites. Indeed, flurbiprofen (a NSAID) has been used as a ligand for affinity chromatography in the purification of PGH synthase. After initial solubilization, the enzyme is passed over an affinity column, in which flurbiprofen was attached to CNBr activated Sepharose 4B through a 3,3'-diaminodipropylamine side arm. PGH synthase activity was retarded, but could not be eluted in a specific manner by NSAIDs. Elution from the column was not improved by substituting more potent NSAIDs. Elution could be achieved by high salt in the absence of NSAIDs or by flufenamic acid, to give a 15-fold enrichment of the PGH synthase activity (Prostaglandins, 10, 983-990, (1975)). This method of flurbiprofen affinity chromatography has not been used routinely in published purification procedures for the synthase. Coupling of the affinity labeling NSAID analogs described herein to a suitably activated solid matrix support such as glass, silica, agarose, sepharose, etc., as could be achieved through methods known in the art, would be expected to provide a substantial improvement in affinity chromatography, since the corresponding parent NSAIDs have a greater affinity for PGH synthase than flurbiprofen. Many enzymes and receptors are commonly purified through the use of competitive ligands bound to affinity resins. This method can greatly reduce the number of steps and time involved in their purification. (For general reviews see J. Biol. Chem., 245, 3059 (1970), and Venter and Harrison, Eds., "Receptor Purification Procedures" in Receptor Biochemistry and Methodology (Alan R. Liss, New York, 1984), the disclosures of which are hereby incorporated by reference).
Affinity labeling agents based on the potent NSAIDs described herein also have the potential to act as pro-drugs. It is generally accepted that aspirin (acetyl salicylate) is rapidly de-acetylated in vivo by plasma esterases to yield free salicylate. The plasma t.sub.178 life of aspirin is 15 minutes. In the treatment of arthritis it is the plasma level of salicylate that is the indicator of therapeutic efficacy. Slow hydrolysis of bromoacetylated analogs of indomethacin, N-phenylanthranilic acids and tolmetin analogs may prolong the half-life of the active drug in the treatment of the arthritic. Some of the therapeutic efficacy of aspirin has also been attributed to its irreversible acetylation of PGH synthase (e.g. platelet PGH synthase). Since the bromoacetylating agents described herein could also irreversibly inactivate PGH synthase in vivo, they may represent superior "aspirin-like" drugs.
Therefore, the affinity labeling analogs of NSAIDs described herein could have a number of utilities: (a) they may act as affinity labeling agents of putative target enzymes e.g. 3.alpha.-HSD/PGH synthase and prostaglandin dehydrogenases, and permit the topography of the NSAID binding site to be mapped; (b) they may act as affinity ligands on immobilized supports for the purification of prostaglandin transforming enzymes and NSAID receptors; (c) by de-acetylation they may act as pro-drugs and (d) by acetylating PGH synthase in vivo they may act as superior "aspirin-like" drugs.